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Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing

BACKGROUND: Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utilit...

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Autores principales: Pitt, Miranda E, Nguyen, Son H, Duarte, Tânia P S, Teng, Haotian, Blaskovich, Mark A T, Cooper, Matthew A, Coin, Lachlan J M
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998412/
https://www.ncbi.nlm.nih.gov/pubmed/32016399
http://dx.doi.org/10.1093/gigascience/giaa002
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author Pitt, Miranda E
Nguyen, Son H
Duarte, Tânia P S
Teng, Haotian
Blaskovich, Mark A T
Cooper, Matthew A
Coin, Lachlan J M
author_facet Pitt, Miranda E
Nguyen, Son H
Duarte, Tânia P S
Teng, Haotian
Blaskovich, Mark A T
Cooper, Matthew A
Coin, Lachlan J M
author_sort Pitt, Miranda E
collection PubMed
description BACKGROUND: Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR). RESULTS: The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network–based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with ∼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, β-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes. CONCLUSION: Overall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.
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spelling pubmed-69984122020-02-10 Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing Pitt, Miranda E Nguyen, Son H Duarte, Tânia P S Teng, Haotian Blaskovich, Mark A T Cooper, Matthew A Coin, Lachlan J M Gigascience Research BACKGROUND: Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR). RESULTS: The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network–based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with ∼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, β-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes. CONCLUSION: Overall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes. Oxford University Press 2020-02-04 /pmc/articles/PMC6998412/ /pubmed/32016399 http://dx.doi.org/10.1093/gigascience/giaa002 Text en © The Author(s) 2020. Published by Oxford University Press. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Pitt, Miranda E
Nguyen, Son H
Duarte, Tânia P S
Teng, Haotian
Blaskovich, Mark A T
Cooper, Matthew A
Coin, Lachlan J M
Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing
title Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing
title_full Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing
title_fullStr Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing
title_full_unstemmed Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing
title_short Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing
title_sort evaluating the genome and resistome of extensively drug-resistant klebsiella pneumoniae using native dna and rna nanopore sequencing
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998412/
https://www.ncbi.nlm.nih.gov/pubmed/32016399
http://dx.doi.org/10.1093/gigascience/giaa002
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